Bispecific antibodies and uses thereof

Abstract

The invention discloses a bispecific antibody and application thereof, and belongs to the technical field of biological medicine. The bispecific antibody comprises one or more first antigen binding regions, one or more second antigen binding regions and one or more third antigen binding regions, wherein the first antigen binding regions have NKp46 binding activity; the first antigen binding region has HLAG binding activity, the second antigen binding region has HLAG binding activity, the first antigen binding region is connected with the second antigen binding region, the second antigen binding region comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and the HCDR1, the HCDR2 and the HCDR3 are respectively selected from SEQ ID NO: 7-9 or amino acid sequences with at least 80% homology with the SEQ ID NO: 7-9. The bispecific antibody disclosed by the invention can target HLAG and NKp46 at the same time, and particularly can be combined with NKp46 on the surface of an NK cell and HLAG on the surface of a tumor cell at the same time, so that the NK cell is directly activated, HLAG positive tumor cells are killed, and HLAG-mediated related diseases can be effectively treated.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to bispecific antibodies and their uses, and more specifically to a bispecific antibody against NKp46 and against HLAG and its uses. Background Technology

[0002] Bispecific antibodies are antibodies that can bind specifically to two antigen sites simultaneously.

[0003] HLAG is a non-classical MHC class I molecule, primarily expressed in placental tissue, with little or no expression in other normal tissues. However, it is highly expressed in many tumor types, such as breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, and renal cell carcinoma, and is negatively correlated with patient prognosis. Therefore, HLAG is a highly desirable tumor target. However, due to the high polymorphism and homology of the HLA family, most antibodies lack true specific HLA-G binding properties.

[0004] NK cells are a very important type of innate immune cell that can recognize and kill virus-infected or malignant tumor cells. NKp46 is a very important activating receptor on the surface of NK cells. Cross-linking NKp46 can promote NK cell activation and degranulation, thereby achieving the killing of tumor cells.

[0005] Currently, there are few bispecific antibodies targeting HLAG and NK cells, and those that exist have low binding specificity. Therefore, developing bispecific antibodies that specifically target HLAG and NK cells has significant clinical implications. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a bispecific antibody that can simultaneously target HLAG and NKp46, particularly capable of simultaneously binding to NKp46 on the surface of NK cells and HLAG on the surface of tumor cells, thereby directly activating NK cells and killing HLAG-positive tumor cells.

[0007] In a first aspect, this application proposes a bispecific antibody. According to an embodiment of this application, the bispecific antibody comprises: one or more first antigen-binding regions having NKp46 binding activity; and one or more second antigen-binding regions having HLAG binding activity, the first and second antigen-binding regions being linked; wherein the second antigen-binding region comprises: heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are respectively selected from SEQ ID NO:7-9 or amino acid sequences having at least 80% homology with SEQ ID NO:7-9. The bispecific antibody of this application can simultaneously target HLAG and NKp46, particularly capable of simultaneously binding to NKp46 on the surface of NK cells and HLAG on the surface of tumor cells, thereby directly activating NK cells and killing HLAG-positive tumor cells.

[0008] In a second aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the bispecific antibody described in the first aspect. The nucleic acid molecule according to embodiments of this application may encode a bispecific antibody capable of simultaneously targeting NKp46 and HLAG.

[0009] In a third aspect, this application provides an expression vector. According to embodiments of this application, the expression vector comprises the nucleic acid molecule described in the second aspect. Thus, the bispecific antibody described in the first aspect can be effectively expressed using the constructed expression vector.

[0010] In a fourth aspect, this application provides a recombinant cell. According to embodiments of this application, the recombinant cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect; or expresses the bispecific antibody described in the first aspect. According to embodiments of this application, the recombinant cell can be obtained by transfection or transformation of the expression vector described in the third aspect, and under suitable conditions can efficiently express the bispecific antibody described in the first aspect.

[0011] In a fifth aspect of this application, a pharmaceutical composition is proposed. According to embodiments of this application, the pharmaceutical composition comprises: the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression method described in the third aspect, or the recombinant cells described in the fourth aspect. As is known prior art, the bispecific antibody described in the first aspect, or the bispecific antibody prepared using the nucleic acid molecule described in the second aspect, the expression method described in the third aspect, or the recombinant cells described in the fourth aspect, can simultaneously target NKp46 and HLAG, particularly binding simultaneously to NKp46 on the surface of NK cells and HLAG on the surface of tumor cells, thereby directly activating NK cells and effectively promoting NK cell killing of tumor cells, exhibiting better anti-cancer activity. Therefore, the obtained pharmaceutical composition can effectively prevent and / or treat HLAG-mediated diseases.

[0012] In a sixth aspect of this application, a kit is provided. According to embodiments of this application, the kit comprises the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect. According to embodiments of this application, the kit can bind to NKp46 protein and / or HLAG protein, and can effectively identify NKp46 protein and / or HLAG protein.

[0013] In a seventh aspect of this application, the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect in the preparation of a kit for detecting NKp46 and / or HLAG. As is known from the foregoing, the bispecific antibody described in the first aspect, or the bispecific antibody prepared using the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect, can simultaneously target NKp46 and HLAG. Therefore, the method of this application can effectively identify NKp46 protein and / or HLAG protein.

[0014] In an eighth aspect of this application, the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a drug for the prevention and / or treatment of HLAG-mediated diseases. As is known from the foregoing, the bispecific antibody described in the first aspect, or the bispecific antibody prepared using the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect, can effectively prevent and / or treat HLAG-mediated diseases. Therefore, the bispecific antibody and the corresponding nucleic acid, vector, or transformant or pharmaceutical composition of this application can be further used to prepare a drug that can be clinically used for the prevention or treatment of HLAG-mediated diseases.

[0015] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0016] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0017] Figure 1 This is a schematic diagram of the bispecific antibody configuration described in the embodiments of this application;

[0018] Figure 2 This is a schematic diagram showing the detection results of the binding ability of the bispecific antibody to HLAG and NKp46 proteins described in the embodiments of this application;

[0019] Figure 3 This is a schematic diagram showing the detection results of the binding ability of the bispecific antibody described in the embodiments of this application to SK-OV-3-human HLAG cells;

[0020] Figure 4 This is a schematic diagram showing the detection results of the binding ability of the bispecific antibody to human primary NK cells described in the embodiments of this application;

[0021] Figure 5 This is a schematic diagram illustrating the detection results of the bispecific antibody promoting human NK cell degranulation as described in the embodiments of this application;

[0022] Figure 6 This is a schematic diagram illustrating the detection results of the bispecific antibody promoting the killing of HLAG-positive cell lines by human PBMCs as described in the embodiments of this application. Detailed Implementation

[0023] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0024] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0025] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. Specifically, A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (valine).

[0026] In this article, the term "bispecific antibody" generally refers to an antibody that recognizes two antigenic epitopes, including but not limited to chimeric antibodies, humanized antibodies, or dimers or multimers, and also includes antibody fragments, as long as they exhibit the desired biological activity. Antibodies can be murine, human, humanized, chimeric, or derived from other species.

[0027] In this article, the "bispecific antibody" contains two antigen-binding fragments, each targeting a different antigen. Each antigen-binding fragment contains a heavy chain variable region, or both a heavy chain variable region and a light chain variable region. Within the variable regions, certain areas exhibit a higher degree of variation in amino acid composition and sequence, termed "hypervariable region (HVR)." The hypervariable region is the site where the antigen and antibody bind, and is therefore also called the complementarity-determining region (CDR). The heavy chain complementarity determining regions (heavy chain variable regions CDRs) are denoted by "HCDRs" or "HCDR", including HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2), and HCDR3 (also known as CDR-H3); the light chain complementarity determining regions (light chain variable regions CDRs) are denoted by "LCDRs" or "LCDR", including LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2), and LCDR3 (also known as CDR-L3). Commonly used CDR definition schemes in this field include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition.

[0028] In this article, the terms "anti-NKp46 antibody" or "antibody that binds to NKp46" refer to antibodies capable of binding to NKp46. The terms "anti-HLAG antibody" or "antibody that binds to HLAG" refer to antibodies capable of binding to HLAG.

[0029] In this article, the term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region of a monoclonal antibody from one species (such as a mouse) with the constant region of an antibody from another species (such as a human).

[0030] In this article, the term "humanized antibody" refers to a chimeric antibody, specifically a recombinant antibody obtained by replacing all non-CDR (Fv backbone region (FR)) amino acid sequences in the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with non-CDR amino acid sequences in the constant and variable regions of an antibody from another species (e.g., human). In other words, when the constant region of an antibody is humanized, it is called a chimeric antibody, and when all non-CDR amino acid sequences in both the constant and variable regions are humanized, it is called a humanized antibody. The humanization method can be performed using conventional antibody engineering techniques, and will not be elaborated upon here.

[0031] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, containing a nucleotide sequence that expresses a target protein, and capable of being transferred to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0032] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., expression vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of this application and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0033] In this document, the term "pharmaceutical composition" generally refers to a composition in unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining an active bispecific antibody with a liquid carrier, a finely fragmented solid carrier, or both.

[0034] In this article, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0035] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients is also within the scope of consideration for this application, except for any range of incompatibilities with the bispecific antibody of this application, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner.

[0036] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The bispecific antibody or pharmaceutical composition of this application may be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but this application is not limited to these exemplified routes of administration. Preferably, the bispecific antibody or pharmaceutical composition of this application is administered via intravenous or subcutaneous injection.

[0037] In this document, the term "treatment" refers to the attainment of a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any medication that administers a drug or bispecific antibody to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administering a drug containing the bispecific antibody described herein to an individual in need.

[0038] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0039] The amino acid sequences involved in this application are shown in Table 1.

[0040] This application discloses a bispecific antibody against NKp46 and against HLAG, a nucleic acid molecule, an expression vector, recombinant cells, a pharmaceutical composition, a kit, and their uses, which will be described in detail below.

[0041] Bispecific antibodies

[0042] In one aspect of this application, a bispecific antibody is proposed. According to an embodiment of this application, the bispecific antibody comprises: one or more first antigen-binding regions having NKp46 binding activity; and one or more second antigen-binding regions having HLAG binding activity, the first and second antigen-binding regions being linked together. The second antigen-binding region includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NO:7-9 or amino acid sequences having at least 80% homology to SEQ ID NO:7-9. The bispecific antibody of this application can simultaneously target HLAG and NKp46, particularly binding simultaneously to NKp46 on the surface of NK cells and HLAG on the surface of tumor cells, thereby directly activating NK cells and killing HLAG-positive tumor cells, effectively treating HLAG-mediated diseases (e.g., cancer).

[0043] According to embodiments of this application, the above-mentioned bispecific antibody may further include at least one of the following technical features:

[0044] In an optional embodiment of this application, both the first antigen-binding region and the second antigen-binding region are antigen-binding fragments.

[0045] In this document, the term "antigen-binding fragment" is equivalent to "antigen-binding antibody fragment" and can include a portion of a complete antibody, generally an antigen-binding region or a variable region. This includes, but is not limited to: Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, scFab (single chain Fab fragment), linear antibodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol ("PEGylated, PEGylated") (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol).

[0046] According to embodiments of this application, the first antigen-binding region is selected from the scFab fragment or the scFv fragment.

[0047] According to embodiments of this application, the first antigen-binding region includes HCDRs and LCDRs, wherein the HCDRs and / or LCDRs are defined by Kabat, Chothia, AbM, Contact, or IMGT.

[0048] Unless otherwise specified, the amino acid sequences of HCDRs and LCDRs in this paper are defined using Kabat.

[0049] According to an embodiment of this application, the first antigen-binding region includes: HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:1 to 3, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:4 to 6, respectively.

[0050] Unless otherwise specified herein, “amino acid sequence as shown in SEQ ID NO:N” or “X as shown in SEQ ID NO:N” includes the amino acid sequence shown in SEQ ID NO:N, an amino acid sequence having a modified form of the amino acid sequence shown in SEQ ID NO:N, and an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:N.

[0051] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0052] In this document, the term "conservatively modified amino acid sequence" refers to an amino acid modification that does not significantly affect or alter the binding properties of an antibody containing that amino acid sequence. Such modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this application using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0053] In this document, without substantially affecting antibody activity (retaining at least 95% of the activity), those skilled in the art may substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequence of this application to obtain variants of the antibody or its functional fragment sequence. These are all considered to be included within the scope of protection of this application. For example, amino acids with similar properties may be substituted in the variable region. The variant sequence described in this application may have at least 80% identity (or homology) with the reference sequence, meaning at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with each reference sequence. The sequence identity described in this application can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this application are shown from the N-terminus to the C-terminus.

[0054] According to embodiments of this application, at least a portion of the framework regions of the light chain variable region and the heavy chain variable region in the scFv fragment are each independently derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies and human antibodies.

[0055] According to embodiments of this application, at least a portion of the framework regions of the light chain variable region and the heavy chain variable region in the scFab fragment are each independently derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies and human antibodies.

[0056] According to embodiments of this application, the first antigen-binding region has a heavy chain variable region as shown in the amino acid sequence SEQ ID NO:13 and a light chain variable region as shown in the amino acid sequence SEQ ID NO:14. In an optional embodiment of this application, the scFab fragment has a first heavy chain variable region as shown in the amino acid sequence SEQ ID NO:13 and a light chain variable region as shown in the amino acid sequence SEQ ID NO:14. In an optional embodiment of this application, the scFv fragment has a first heavy chain variable region as shown in the amino acid sequence SEQ ID NO:13 and a light chain variable region as shown in the amino acid sequence SEQ ID NO:14.

[0057] According to an embodiment of this application, the first antigen-binding region is selected from the scFab fragment, wherein the CH1 fragment and the CL fragment in the scFab fragment are each independently derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

[0058] According to an embodiment of this application, the first antigen-binding region is selected from the scFab fragment, and the CH1 fragment in the scFab fragment is selected from the CH1 fragment of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0059] It should be noted that the immunoglobulins discussed in this article can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins can originate from any species.

[0060] According to embodiments of this application, the CL fragment in the scFab fragment is selected from κ-type or λ-type CL fragments.

[0061] According to an embodiment of this application, the first antigen-binding region is selected from the scFab fragment, which has an amino acid sequence as shown in SEQ ID NO:17.

[0062] According to an embodiment of this application, the first antigen-binding region is selected from the scFv fragment, which has an amino acid sequence as shown in SEQ ID NO:18.

[0063] According to embodiments of this application, the second antigen-binding region is selected from the Fab fragment.

[0064] Those skilled in the art will know that Fab antibodies consist of two chains: a heavy chain variable region + CH1 and a light chain variable region + CL (i.e., a light chain constant region).

[0065] According to embodiments of this application, the second antigen-binding region includes HCDRs and LCDRs, wherein the HCDRs and / or LCDRs are defined by Kabat, Chothia, AbM, Contact, or IMGT.

[0066] According to an embodiment of this application, the second antigen-binding region includes: HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:7-9, and LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:10-12.

[0067] According to embodiments of this application, at least a portion of the framework regions of the light chain variable region and the heavy chain variable region in the Fab fragment are each independently derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies and human antibodies.

[0068] According to embodiments of this application, the second antigen-binding region has a heavy chain variable region as shown in the amino acid sequence SEQ ID NO:15 and a second light chain variable region as shown in the amino acid sequence SEQ ID NO:16. In an optional embodiment of this application, the Fab fragment has a second heavy chain variable region as shown in the amino acid sequence SEQ ID NO:15 and a second light chain variable region as shown in the amino acid sequence SEQ ID NO:16.

[0069] According to an embodiment of this application, the second antigen-binding region is selected from the Fab fragment, wherein the CH1 fragment and the CL fragment in the Fab fragment are each independently derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

[0070] According to an embodiment of this application, the second antigen-binding region is selected from the Fab fragment, wherein the CH1 fragment of the Fab fragment is selected from the CH1 fragment of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0071] According to embodiments of this application, the CL fragment in the Fab fragment is selected from κ-type or λ-type CL fragments.

[0072] According to an embodiment of this application, the second antigen-binding region is selected from the Fab fragment, one chain of which has an amino acid sequence as shown in SEQ ID NO:19 and the other chain has an amino acid sequence as shown in SEQ ID NO:20.

[0073] According to an embodiment of this application, the bispecific antibody further includes an Fc fragment, at least one first antigen-binding region is linked to the Fc fragment, and at least one second antigen-binding region is linked to the Fc fragment.

[0074] According to embodiments of this application, the Fc fragment is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably a human Fc fragment.

[0075] According to embodiments of this application, the Fc fragment is derived from the Fc fragment of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0076] According to embodiments of this application, the Fc fragment is a human IgG1 Fc fragment or a mutant thereof.

[0077] According to embodiments of this application, the bispecific antibody has a symmetrical or asymmetrical structure.

[0078] According to an embodiment of this application, the bispecific antibody has a symmetrical structure, and compared with the wild-type human IgG1 Fc fragment, the mutant of the human IgG1 Fc fragment has L234A and L235A mutations.

[0079] According to an embodiment of this application, the bispecific antibody has an asymmetric structure, and the two chains of the Fc fragment are connected by a knock-in-hole structure.

[0080] In one embodiment of this application, the "knob into hole structure" is formed by creating a button (hole) mutation in the CH3 region of the constant region of the antibody heavy chain, which facilitates heavy chain interlocking and the formation of a heterodimer. For example, this can be achieved by mutating the amino acids in the CH3 domain of the constant region of the human IgG1 heavy chain (one chain has T366S, L368A, Y407V, Y349C mutations, i.e., "hole"; the other chain has T366W, S354C mutations, i.e., "knob").

[0081] According to an embodiment of this application, the bispecific antibody has a symmetrical structure and includes at least two first antigen-binding regions.

[0082] According to an embodiment of this application, the bispecific antibody has a symmetrical structure and includes at least two second antigen-binding regions.

[0083] According to an embodiment of this application, the bispecific antibody has a symmetrical structure, with the first antigen-binding region connected to the C-terminus of the Fc fragment and the second antigen-binding region connected to the N-terminus of the Fc fragment.

[0084] According to an embodiment of this application, the bispecific antibody has a symmetrical structure, and the first antigen-binding region is a scFv fragment.

[0085] According to an embodiment of this application, the bispecific antibody has a symmetrical structure and includes a first linker peptide.

[0086] According to an embodiment of this application, the N-terminus of the first antigen-binding region is connected to the C-terminus of the first linker peptide, and the N-terminus of the first linker peptide is connected to the C-terminus of the Fc fragment.

[0087] In a preferred embodiment of this application, see this application. Figure 1The D structure of the scFv fragment has the N-terminus linked to the C-terminus of the first linker peptide, the N-terminus of the first linker peptide being linked to the C-terminus of the Fc fragment, and the N-terminus of the Fc fragment being linked to the C-terminus of CH1 in the Fab fragment.

[0088] According to an embodiment of this application, the first linker peptide has an amino acid sequence as shown in (GGGGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0089] According to embodiments of this application, the first linker peptide has a structure such as (GGGGS)3, (GGGGS)4, or (GGGGS) 12 The amino acid sequence shown.

[0090] According to an embodiment of this application, the bispecific antibody has an asymmetric structure and includes a first antigen-binding region.

[0091] According to embodiments of this application, the bispecific antibody has an asymmetric structure, and the bispecific antibody includes one or two second antigen-binding regions.

[0092] According to an embodiment of this application, the bispecific antibody has an asymmetric structure, wherein the first antigen-binding region is connected to the N-terminus of one chain in the Fc fragment, and the second antigen-binding region is connected to the N-terminus of the other chain in the Fc fragment.

[0093] According to an embodiment of this application, the bispecific antibody has an asymmetric structure, the first antigen-binding region is an scFv fragment, and the second antigen-binding region is selected from two fragments, both of which are Fab fragments.

[0094] It should be noted that when the first antigen-binding region is an antigen-binding fragment containing two chains (e.g., Fab), it is preferable to link the peptide chain containing the heavy chain variable region to the Fc fragment.

[0095] According to an embodiment of this application, the first antigen-binding region is connected to the N-terminus of one chain in the Fc fragment, one second antigen-binding region is connected to the N-terminus of another chain in the Fc fragment, and another second antigen-binding region is connected to the N-terminus of the first antigen-binding region.

[0096] In a preferred embodiment of this application, see this application. Figure 1 The A structure has the C-terminus of the scFv fragment connected to the N-terminus of one chain in the Fc fragment, the C-terminus of CH1 in one Fab fragment connected to the N-terminus of another chain in the Fc fragment, and the C-terminus of CH1 in another Fab fragment connected to the N-terminus of the first antigen-binding region.

[0097] According to the embodiments of this application, see this application. Figure 1 In the A structure, the C-terminus of the light chain variable region in the scFv segment is connected to the N-terminus of the heavy chain variable region in the scFv segment.

[0098] According to an embodiment of this application, the bispecific antibody has an asymmetric structure, the first antigen-binding region is an scFv fragment, and the second antigen-binding region is selected from one of the Fab fragments.

[0099] According to the embodiments of this application, see this application. Figure 1 The B structure is such that the C end of the light chain variable region in the scFv segment is connected to the N end of the heavy chain variable region in the scFv segment.

[0100] According to an embodiment of this application, the bispecific antibody has an asymmetric structure, the first antigen-binding region is an scFab fragment, and the second antigen-binding region is selected from one of the scFab fragments.

[0101] According to the embodiments of this application, see this application. Figure 1 The C-structure is such that the N-terminus of the heavy chain variable region in the scFab segment is connected to the C-terminus of the light chain variable region in the scFab segment.

[0102] According to embodiments of this application, the bispecific antibody comprises:

[0103] Having a first polypeptide chain as shown in the amino acid sequence of SEQ ID NO:20 and a second polypeptide chain as shown in the amino acid sequence of SEQ ID NO:21; or

[0104] Having a first polypeptide chain as shown in the amino acid sequence SEQ ID NO:20, a second polypeptide chain as shown in the amino acid sequence SEQ ID NO:22, a third polypeptide chain as shown in the amino acid sequence SEQ ID NO:23, and a fourth polypeptide chain as shown in the amino acid sequence SEQ ID NO:20; or

[0105] Having a first polypeptide chain as shown in the amino acid sequence of SEQ ID NO:20, a second polypeptide chain as shown in the amino acid sequence of SEQ ID NO:22, and a third polypeptide chain as shown in the amino acid sequence of SEQ ID NO:24; or

[0106] It has a first polypeptide chain as shown in the amino acid sequence of SEQ ID NO:20, a second polypeptide chain as shown in the amino acid sequence of SEQ ID NO:22, and a third polypeptide chain as shown in the amino acid sequence of SEQ ID NO:25.

[0107] Nucleic acid molecules, expression vectors and recombinant cells

[0108] In a second aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the bispecific antibody described in the first aspect. The nucleic acid molecule according to embodiments of this application may encode a bispecific antibody capable of simultaneously targeting NKp46 and HLAG.

[0109] According to an embodiment of this application, the nucleic acid molecule is DNA.

[0110] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0111] Those skilled in the art will understand that the features and advantages described above for bispecific antibodies also apply to this nucleic acid molecule, and will not be repeated here.

[0112] In a third aspect, this application provides an expression vector. According to embodiments of this application, the expression vector includes the nucleic acid molecule described in the second aspect. When linking the nucleic acid molecule to the expression vector, the nucleic acid molecule can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the expression vector itself. Naturally, the nucleic acid molecule and the control elements only need to be operably linked.

[0113] In this article, "operable ligation" refers to ligating a foreign gene into an expression vector so that the control elements within the expression vector, such as transcriptional and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors include plasmids and bacteriophages.

[0114] According to some specific embodiments of this application, after the expression vector is introduced into suitable recipient cells, the aforementioned bispecific antibody can be effectively expressed under the mediation of a regulatory system, thereby achieving the large-scale in vitro production of bispecific antibodies.

[0115] According to embodiments of this application, the expression vector may refer to a cloning vector, which can be obtained by operatively ligating the nucleic acid molecule to a commercially available vector (such as a plasmid or viral vector). The vector used in this application is not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0116] In this document, the term "operably ligated" refers to ligating a foreign gene to an expression vector such that the control elements within the expression vector, such as transcriptional control amino acid sequences and translational control amino acid sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors include viral vectors, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of this application is introduced into a suitable host cell, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the in vitro large-scale production of bispecific antibodies encoded by the nucleic acid molecules.

[0117] According to embodiments of this application, the carrier is a eukaryotic carrier or a prokaryotic carrier.

[0118] According to embodiments of this application, the vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0119] Those skilled in the art will understand that the features and advantages described above for bispecific antibodies and nucleic acid molecules also apply to this expression vector, and will not be repeated here.

[0120] In a fourth aspect, this application provides a recombinant cell. According to embodiments of this application, the recombinant cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect; or expresses the bispecific antibody described in the first aspect. According to embodiments of this application, the recombinant cell can be obtained by transfection or transformation of the expression vector described in the third aspect, and under suitable conditions can efficiently express the bispecific antibody described in the first aspect.

[0121] According to some specific embodiments of this application, the recombinant cells can efficiently and extensively express bispecific antibodies under suitable conditions. The bispecific antibodies have stronger specificity, longer half-life and higher potency, and can deliver antibody drugs to target cells with a smaller drug dose, thereby achieving effective treatment or prevention of HLAG-mediated diseases. They have low toxicity and higher safety.

[0122] It should be noted that "suitable conditions" refers to conditions suitable for the expression of the bispecific antibody described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the bispecific antibody include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the above-mentioned conditions for the expression of the bispecific antibody according to the specific environment of their laboratory.

[0123] According to embodiments of this application, the recombinant cells are obtained by introducing the expression vector described in the third aspect into host cells.

[0124] It should be noted that the recombinant cells described in this application are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, and Trichoderma; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments of this application, the recombinant cells are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0125] Those skilled in the art will understand that the features and advantages described above for bispecific antibodies, nucleic acid molecules and expression vectors also apply to this recombinant cell, and will not be repeated here.

[0126] Pharmaceutical Compositions and Kits

[0127] In a fifth aspect of this application, a pharmaceutical composition is provided. According to embodiments of this application, the pharmaceutical composition comprises: the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression method described in the third aspect, or the recombinant cells described in the fourth aspect. It is known that the bispecific antibody described in the first aspect, or the bispecific antibody prepared using the nucleic acid molecule described in the second aspect, the expression method described in the third aspect, or the recombinant cells described in the fourth aspect, can simultaneously target NKp46 and HLAG, effectively promoting NK cell killing of tumor cells and exhibiting better anti-cancer activity. Therefore, the obtained pharmaceutical composition can effectively prevent and / or treat HLAG-mediated diseases.

[0128] According to embodiments of this application, pharmaceutically acceptable excipients are further included.

[0129] According to embodiments of this application, the excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

[0130] It should be noted that the pharmaceutical composition includes combinations that are separate in time and / or space, as long as they can work together to achieve the purpose of this application. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0131] According to embodiments of this application, the pharmaceutical composition of this application contains a safe and effective amount of the active ingredient of this application and pharmaceutically acceptable excipients.

[0132] The effective amount of the active ingredient described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0133] Those skilled in the art will understand that the features and advantages described above for bispecific antibodies, nucleic acid molecules, expression vectors and recombinant cells also apply to this pharmaceutical composition, and will not be repeated here.

[0134] In a sixth aspect of this application, a kit is provided. According to embodiments of this application, the kit comprises the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect. According to embodiments of this application, the kit can bind to NKp46 protein and / or HLAG protein, and can effectively identify NKp46 protein and / or HLAG protein.

[0135] As mentioned above, the bispecific antibody in this application can specifically bind to NKp46 and HLAG. The NKp46 protein and / or HLAG protein related kits developed using this property can be used for NKp46 protein and / or HLAG protein related research, such as for detecting and / or enriching and / or separating and purifying human or other mammalian NKp46 protein and / or HLAG protein.

[0136] The kit described herein can effectively detect, enrich, or separate and purify NKp46 and / or HLAG proteins in biological samples for further scientific research, such as qualitative or quantitative detection of NKp46 and / or HLAG protein molecules in biological samples. More specifically, it can be used in kits involving the specific binding properties of NKp46 and / or HLAG proteins to bispecific antibodies, such as immunoblotting and immunoprecipitation. These kits, in addition to containing the bispecific antibody of this application, may also contain any one or more of the following: protein purification columns; immunoglobulin affinity purification buffers; and cell assay diluents. The bispecific antibody of this application can be used for different types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, it can be used to test for HLAG-mediated diseases by detecting the serum or blood of a subject.

[0137] Those skilled in the art will understand that the features and advantages described above for bispecific antibodies, nucleic acid molecules, expression vectors and recombinant cells also apply to this kit, and will not be repeated here.

[0138] use

[0139] In a seventh aspect of this application, the invention discloses the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect in the preparation of a kit for detecting NKp46 and / or HLAG. As is known from the foregoing, the bispecific antibody described in the first aspect, or the bispecific antibody prepared using the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect, can simultaneously target NKp46 and HLAG. Therefore, the method of this application can effectively identify NKp46 protein and / or HLAG protein.

[0140] As previously described, the bispecific antibody of this application can specifically bind to NKp46 and HLAG. Therefore, the bispecific antibody can be used to detect NKp46 and / or HLAG. Furthermore, it can be used to prepare NKp46 and / or HLAG-related kits for scientific research, such as qualitative or quantitative detection of NKp46 and / or HLAG protein molecules in biological samples. More specifically, it can be used in kits involving the specific binding properties of NKp46 and / or HLAG to antibodies, such as immunoblotting and immunoprecipitation. These kits, in addition to containing the bispecific antibody of this application, may also contain any one or more of the following: antagonists; protein purification columns; immunoglobulin affinity purification buffers; and cell assay diluents. The bispecific antibody of this application can be used for different types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, it can be used to test for HLAG-mediated diseases by detecting the serum or blood of a subject.

[0141] Those skilled in the art will understand that the features and advantages described above for bispecific antibodies, nucleic acid molecules, expression vectors and recombinant cells also apply to this use, and will not be repeated here.

[0142] In an eighth aspect of this application, the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a drug for the prevention and / or treatment of HLAG-mediated diseases. It is known that the bispecific antibody described in the first aspect, or the bispecific antibody prepared using the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect, can effectively prevent and / or treat HLAG-mediated diseases. Therefore, the bispecific antibody and the corresponding nucleic acid, vector, or transformant or pharmaceutical composition of this application can be further used to prepare a drug that can be clinically used for the prevention or treatment of HLAG-mediated diseases.

[0143] According to embodiments of this application, the HLAG-mediated diseases include cancer, preferably HLAG-positive cancer.

[0144] According to embodiments of this application, the cancer includes at least one of choriocarcinoma, esophageal cancer, head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, bile duct cancer, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

[0145] Those skilled in the art will understand that the features and advantages described above for bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells and pharmaceutical compositions are equally applicable to this use and will not be repeated here.

[0146] method

[0147] In a ninth aspect of this application, a method for preventing and / or treating NKp46 and / or HLAG-mediated diseases is provided. According to embodiments of this application, the method comprises administering to a subject a pharmaceutically acceptable amount of the bispecific antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cells of the fourth aspect, or the pharmaceutical composition of the fifth aspect.

[0148] It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one implementation, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0149] The effective amount of the bispecific antibody, nucleic acid molecule, expression vector, recombinant cell, or pharmaceutical composition described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0150] According to embodiments of this application, the HLAG-mediated diseases include cancer.

[0151] According to embodiments of this application, the cancer includes at least one of choriocarcinoma, esophageal cancer, head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, bile duct cancer, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

[0152] Those skilled in the art will understand that the features and advantages described above for bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, and pharmaceutical compositions are equally applicable to this method and will not be repeated here.

[0153] The amino acid and nucleotide sequences in this application are shown in Table 1:

[0154] Table 1

[0155]

[0156]

[0157]

[0158] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0159] Example 1: Design and construction of NKp46-HLAG bispecific antibody

[0160] In this embodiment, four different anti-NKp46-anti-HLAG bispecific antibodies were constructed through gene synthesis. Figure 1 The scFv of the anti-NKp46 antibody is linked via linker peptide 1, the scFab via linker peptide 3, and different antibodies are linked via linker peptide 2. The heavy chain of the aforementioned bispecific antibodies uses the human IgG1 subtype. To prevent cross-linking activation caused by the Fcγ receptor, a (L234A / L235A) mutation was also introduced in the Fc segment of the heavy chain. Furthermore, different amino acid mutations were introduced into the constant regions of polypeptide chain 2 (with S354C and T366W mutations) and polypeptide chain 3 (with Y349C, T366S, L368A, and Y407V mutations) to form a knobinhole structure. The sequences of the anti-HLAG antibody, anti-NKp46 antibody, and bispecific antibody are shown in Table 1.

[0161] The above-mentioned polypeptide sequences were constructed into pcDNA3.4 vectors (synthesized by Nanjing GenScript Biotech Co., Ltd.) using molecular cloning technology. The pcDNA3.4 vectors containing the genes encoding bispecific antibody polypeptide chains 1 and 2 were transfected into ExpiCHO-S cells (Gibco) using the ExpiFectamine CHO transfection kit (Gibco). 18-22 h post-transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed and immediately added to the transfected cells. The cells were then incubated at 32°C with 5% CO2 and shaking at 120 rpm. On day 5 post-transfection, 8 mL of ExpiCHO Feed was added to the cells, mixed, and the cells were cultured further. After 10-14 days, the cell culture supernatant was harvested by centrifugation and purified using a Protein A affinity chromatography column (NanoTech) to obtain the bispecific antibodies NAG46-1 (amino acid sequences of the first polypeptide chain as shown in SEQ ID NO:20, the second polypeptide chain as shown in SEQ ID NO:22, the third polypeptide chain as shown in SEQ ID NO:23, and the fourth polypeptide chain as shown in SEQ ID NO:20), NAG46-2 (amino acid sequences of the first polypeptide chain as shown in SEQ ID NO:20, the second polypeptide chain as shown in SEQ ID NO:22, and the third polypeptide chain as shown in SEQ ID NO:24), NAG46-3 (amino acid sequences of the first polypeptide chain as shown in SEQ ID NO:20, the second polypeptide chain as shown in SEQ ID NO:22, and the third polypeptide chain as shown in SEQ ID NO:25), and NAG46-4 (amino acid sequences of the first polypeptide chain as shown in SEQ ID NO:20 and the second polypeptide chain as shown in SEQ ID NO:25). NO:21 shown).

[0162] Example 2: Affinity determination of bispecific antibodies

[0163] The affinity of bispecific antibodies NAG46-1, NAG46-2, and NAG46-4 for HLAG protein (Kaikai Biotechnology) and NKp46 protein (Kaikai Biotechnology) was determined using surface plasmon resonance (SPR) technology. The specific procedure was as follows: The Biacore 1K instrument (Cytiva) was turned on, and the Protein A chip (Cytiva) was placed inside. The chip was then equilibrated in HBS-EP+ buffer. The target antibody at a concentration of 0.5 μg / mL was then flowed through the chip surface at a rate of 30 μL / min to capture the antibody. Different concentrations of HLAG protein or NKp46 protein were then flowed through the chip surface at a rate of 30 μL / min, with a binding time of 90 s, followed by dissociation for 600 s. Kinetic analysis was performed using a 1:1 binding model.

[0164] The affinity values ​​of the bispecific antibodies for human HLAG protein and human NKp46 protein are shown in Table 2.

[0165] Table 2: Results of Bispecific Antibody Affinity Detection

[0166] Bispecific antibodies antigen Ka(1 / Ms) Kd(1 / s) KD(M) NAG46-1 HLAG 1.01E+06 6.03E-04 5.98E-10 NAG46-1 NKp46 1.48E+06 6.01E-04 4.06E-10 NAG46-2 HLAG 1.03E+06 3.58E-04 3.48E-10 NAG46-2 NKp46 1.47E+06 1.23E-03 8.40E-10 NAG46-4 HLAG 1.22E+06 2.71E-04 2.23E-10 NAG46-4 NKp46 6.66E+05 4.56E-04 6.85E-10

[0167] Example 3: Identification of the binding ability of bispecific antibodies to HLAG and NKp46 proteins

[0168] Human NKp46 protein (Kaikai Biotechnology) was diluted to 1 μg / mL with PBS buffer and added to each well in a 96-well plate at a volume of 100 μL. The plate was incubated overnight at 4°C. The PBS buffer in the 96-well plate was discarded. The plate was washed three times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, and then 300 μL / well of 5% BSA was added for blocking. The blocking solution was discarded, and the antibody to be tested was diluted to the appropriate concentration with 100 μL / well of 0.05% BSA. The plate was then incubated at room temperature for 1 hour. After washing three times with PBST, biotinylated human HLAG protein (Kaikai Biotechnology) was diluted to 20 ng / mL with 0.05% BSA and added to each well in a 100 μL solution. The plate was incubated at room temperature for 1 hour. After washing the plate three times with PBST, add 100 μL of horseradish peroxidase (HRP)-labeled streptavidin secondary antibody (Southern Biotech) diluted with 0.5% BSA and incubate at room temperature for 1 h. After washing the plate three times with PBST, add 80 μL / well TMB and incubate at room temperature for 3-5 min. Stop the reaction by adding 80 μL / well stop solution. Read the absorbance at 450 nm using a microplate reader. Specific experimental results are as follows: Figure 2 As shown, the bispecific antibodies NAG46-1, NAG46-2 and NAG46-4 of the present invention can effectively bind to HLAG protein and NKp46 protein.

[0169] Example 4: Identification of the binding ability of bispecific antibodies to SK-OV-3-human HLAG cells

[0170] HEK293T cells were processed at a rate of 5 × 10 5 Seed cells / well in 6-well plates and cultured overnight in DMEM medium without antibiotics. Before transfection, discard the medium and add 1 mL of fresh DMEM medium without antibiotics. Add pLVXEF1ahuman HLA-GIRES-zsGreen (with the inserted nucleotide sequence encoding human HLA-G protein SEQ ID NO:16) to 200 μL of serum-free DMEM medium at a ratio of 2:1:1, along with pMD2G and psPAX2 vector (3 μg total). Then add 12 μg of polyetherimide (PEI, Polysciences Ltd.), mix well, and let stand for 16 min. Add all the liquid to the 6-well plates seeded with HEK293T cells. After culturing for 6 h, discard the medium and add fresh complete DMEM medium. 48 h after transfection, collect the cell culture supernatant and filter through a 0.45 μm filter (Millipore) to obtain lentiviral supernatant. Add all of the obtained lentiviral supernatant to a container containing 1×10⁻⁶ cells / well. 4 Add 4 μg / mL of polybrene (Sigma) to 6-well plates containing either SK-OV-3 cells (ATCC ID: HTB-77) or NCI-H1299 cells (ATCC ID: CRL5803) and incubate for 12 h. Then discard the supernatant and add fresh, complete IMEM medium. The resulting cells are SK-OV-3-HLA-G or NCI-H1299-HLAG.

[0171] SK-OV-3-human HLAG cells were diluted to 1×10⁻⁶ with PBS. 6 Cells were added at a rate of 90 μL / well to a 96-well plate, followed by 10 μL / well of goat serum, and blocked at 4°C for 30 min. A series of serially diluted test antibodies were then added, and the plates were incubated at 4°C for 30 min. After incubation, the cells were washed twice with PBS, followed by the addition of AF647-labeled goat anti-human IgG Fc antibody (Jackson ImmunoResearch), and incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μL / well of PBS and analyzed by flow cytometry. Specific experimental results are shown below. Figure 3 As shown, the bispecific antibodies NAG46-1, NAG46-2 and NAG46-4 of the present invention can effectively bind to SK-OV-3-HLAG cells.

[0172] Example 5: Identification of the binding ability of bispecific antibodies to human primary NK cells

[0173] Dilute PBMC (Miaoshun Biotechnology) to 1×10⁻⁶ with PBS. 6 Cells were added at a rate of 90 μL / well to a 96-well plate, followed by 10 μL / well of goat serum, and blocked at 4°C for 30 min. A series of serially diluted test antibodies were then added, and the plates were incubated at 4°C for 30 min. After incubation, the cells were washed twice with PBS, followed by the addition of AF647-labeled goat anti-human IgG Fc antibody (Jackson Immuno Research) and BV605-CD56 antibody (Biolegend), and incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μL / well of PBS and analyzed by flow cytometry. Specific experimental results are shown below. Figure 4 As shown, the bispecific antibodies NAG46-1, NAG46-2, and NAG46-4 of the present invention can effectively bind to human CD56. + NK cells.

[0174] Example 6: Identification of the ability of bispecific antibodies to promote NK cell activation

[0175] The cultured SK-OV3-HLAG cell line was collected by trypsin digestion and the cell density was adjusted to approximately 1 × 10⁶ cells / year in complete RPMI 1640 medium. 5 PBMCs were added to 96-well plates at a concentration of 50 μL / well. The PBMCs were then diluted to 1.25 × 10⁻⁶ mL with complete RPMI 1640 medium. 6 Cells were added at a rate of 80 μL / well to each of the above 96-well plates, along with 20 μL / well of each of the different concentrations of the target antibody. The plates were incubated at 37°C and 5% CO2 for 18-24 h. After incubation, the cells were washed once with PBS, followed by the addition of BV605-CD56 antibody (Biolegend) and BV421-CD69 antibody (Biolegend), and incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μL / well of PBS and analyzed by flow cytometry. Specific experimental results are shown below. Figure 5 As shown, the bispecific antibodies NAG46-1, NAG46-2, and NAG46-4 of the present invention can effectively promote human CD56 + NK cells express the activation marker CD69.

[0176] Example 7: Identification of the in vitro PBMC-promoting killing ability of bispecific antibodies

[0177] Add 50 μL of complete RPMI 1640 medium to each well of a 96-well RTCA plate and calibrate. Collect cultured HLAG-positive cancer cell lines (e.g., JEG-3 cells, SKOV3-HLAG cells, NCI-H1299-HLAG cells, TE15 cells) using trypsin digestion, and adjust the cell density to approximately 2 × 10⁶ cells using RPMI 1640 medium. 5 Cell counts / mL were individually added to each well of the calibrated RTCA plate at a volume of 50 μL / well, and cell coefficients were measured using an xCELLigence RTCA MP instrument at 37°C and 5% CO2 for 24 h. Subsequently, PBMC (Miaoshun Biotechnology) was diluted to 1.25 × 10⁻⁶ mcg / mL with complete RPMI 1640 medium. 6 Cell counts were increased to 80 μL / well in RTCA plates, along with 20 μL / well of each different concentration of the target antibody. The plates were incubated at 37°C with 5% CO2, and cell coefficients were detected using an xCELLigence RTCA MP instrument. Specific experimental results are shown below. Figure 6 As shown, the anti-NKp46-HLAG bispecific antibodies NAG46-1, NAG46-2 and NAG46-4 of the present invention can effectively promote the killing of HLAG-positive tumor cells by human PBMCs.

[0178] In summary, the experimental results show that the anti-NKp46-HLAG bispecific antibody obtained in this application can bind to HLAG-expressing tumor cells and NK cells expressing NKp46 with high affinity, thereby effectively promoting the killing of HLAG-positive tumor cells by human PBMCs.

[0179] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0180] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A bispecific antibody, characterized in that, include: One or more first antigen-binding regions, wherein the first antigen-binding regions have NKp46 binding activity; One or more second antigen-binding regions, the second antigen-binding regions having HLAG binding activity, and the first antigen-binding regions and the second antigen-binding regions being connected together; The second antigen-binding region includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from the amino acid sequences shown in SEQ ID NO:7-9.

2. The bispecific antibody according to claim 1, characterized in that, The bispecific antibody has at least one of the following characteristics: i) The bispecific antibody further includes an Fc fragment, at least one first antigen-binding region is linked to the Fc fragment, and at least one second antigen-binding region is linked to the Fc fragment; ii) The bispecific antibody has a symmetrical or asymmetrical structure; iii) The first antigen-binding region is selected from the scFab fragment or the scFv fragment; iv) The first antigen-binding region includes HCDRs and LCDRs, wherein the HCDRs and / or LCDRs are defined by Kabat, Chothia, AbM, Contact, or IMGT; v) The first antigen-binding region includes: HCDR1, HCDR2, and HCDR3 are represented by the amino acid sequences of SEQ ID NO:1-3, respectively. LCDR1, LCDR2, and LCDR3 are respectively represented by the amino acid sequences of SEQ ID NO:4-6; vi) At least a portion of the framework regions of the light chain variable region and the heavy chain variable region in the scFv fragment are each independently derived from at least one of the following: mouse antibody, primate antibody, bovine antibody, equine antibody, dairy cow antibody, porcine antibody, sheep antibody, goat antibody, canine antibody, feline antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting rooster antibody, or mutants thereof, preferably at least one of mouse antibody and human antibody; vii) At least a portion of the framework regions of the light chain variable region and the heavy chain variable region in the scFab fragment are each independently derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies and human antibodies; viii) The second antigen-binding region is selected from the Fab fragment; ix) The second antigen-binding region includes HCDRs and LCDRs, wherein the HCDRs and / or LCDRs are defined by Kabat, Chothia, AbM, Contact, or IMGT; x) The second antigen-binding region includes: LCDR1, LCDR2, and LCDR3 are respectively represented by the amino acid sequences SEQ ID NO:10-12; xi) At least a portion of the framework regions of the light chain variable region and the heavy chain variable region in the Fab fragment are each independently derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies and human antibodies.

3. The bispecific antibody according to claim 2, characterized in that, The bispecific antibody has a symmetrical structure and has at least one of the following characteristics: a1) The bispecific antibody comprises at least two first antigen-binding regions; b1) The bispecific antibody comprises at least two second antigen-binding regions; c1) The first antigen-binding region is connected to the C-terminus of the Fc fragment, and the second antigen-binding region is connected to the N-terminus of the Fc fragment; d1) The first antigen-binding region is selected from the scFv fragment; e1) The bispecific antibody includes a first linker peptide; Preferably, the N-terminus of the first antigen-binding region is connected to the C-terminus of the first linker peptide, and the N-terminus of the first linker peptide is connected to the C-terminus of the Fc fragment. Preferably, the first linker peptide has an amino acid sequence as shown in (GGGGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, the first linker peptide has a composition such as (GGGGS)3, (GGGGS)4, or (GGGGS) 12 The amino acid sequence shown.

4. The bispecific antibody according to claim 2, characterized in that, The bispecific antibody has an asymmetric structure and has at least one of the following characteristics: a2) The bispecific antibody includes a first antigen-binding region; b2) The bispecific antibody includes one or two second antigen-binding regions; c2) The first antigen-binding region is connected to the N-terminus of one strand of the Fc fragment, and the second antigen-binding region is connected to the N-terminus of the other strand of the Fc fragment. d2) The two chains of the Fc segment are connected by a knock-into-hole structure.

5. The bispecific antibody according to claim 4, characterized in that, The bispecific antibody has at least one of the following characteristics: e2) The first antigen-binding region is an scFv fragment, and the second antigen-binding region is selected from two, wherein both second antigen-binding regions are Fab fragments; f2) The first antigen-binding region is an scFv fragment, and the second antigen-binding region is selected from one of the Fab fragments; g2) The first antigen-binding region is an scFab fragment, and the second antigen-binding region is selected from one of the Fab fragments; Preferably, the C-terminus of the light chain variable region in the scFv fragment is connected to the N-terminus of the heavy chain variable region in the scFv fragment; Preferably, in e2), the first antigen-binding region is connected to the N-terminus of one chain in the Fc fragment, one second antigen-binding region is connected to the N-terminus of the other chain in the Fc fragment, and another second antigen-binding region is connected to the N-terminus of the first antigen-binding region. Preferably, the N-terminus of the heavy chain variable region in the scFab segment is connected to the C-terminus of the light chain variable region in the scFab segment.

6. The bispecific antibody according to any one of claims 2 to 5, characterized in that, The first antigen-binding region has a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO:13 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO:14; And / or, the first antigen-binding region is selected from the scFab fragment, wherein the CH1 fragment and the CL fragment of the scFab fragment are each independently derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof; And / or, the first antigen-binding region is selected from the scFab fragment, wherein the CH1 fragment of the scFab fragment is selected from the CH1 fragment of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; And / or, the CL fragment in the scFab fragment is selected from κ-type or λ-type CL fragments; And / or, the second antigen-binding region has a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO:15 and a second light chain variable region as shown in the amino acid sequence of SEQ ID NO:16; And / or, the second antigen-binding region is selected from the Fab fragment, wherein the CH1 fragment and the CL fragment of the Fab fragment are each independently derived from at least one of the following: mouse antibody, primate antibody, bovine antibody, equine antibody, dairy bovine antibody, porcine antibody, sheep antibody, goat antibody, canine antibody, feline antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting rooster antibody, or mutants thereof; And / or, the second antigen-binding region is selected from the Fab fragment, wherein the CH1 fragment of the Fab fragment is selected from the CH1 fragment of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; And / or, the CL fragment in the Fab fragment is selected from κ-type or λ-type CL fragments; And / or, the Fc fragment is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably a human Fc fragment; Optionally, the first antigen-binding region is selected from the scFab fragment, which has the amino acid sequence shown in SEQ ID NO:17; Optionally, the first antigen-binding region is selected from the scFv fragment, which has the amino acid sequence shown in SEQ ID NO:18; Optionally, the second antigen-binding region is selected from the Fab fragment, one chain of which has the amino acid sequence shown in SEQ ID NO:19 and the other chain has the amino acid sequence shown in SEQ ID NO:20; Optionally, the Fc fragment is derived from the Fc fragment of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Optionally, the Fc fragment is a human IgG1 Fc fragment or a mutant thereof; Optionally, the bispecific antibody has a symmetrical structure, and the mutant of the human IgG1 Fc fragment has L234A and L235A mutations compared to the wild-type human IgG1 Fc fragment.

7. The bispecific antibody according to claim 1, characterized in that, The bispecific antibody includes: Having a first polypeptide chain as shown in the amino acid sequence of SEQ ID NO:20 and a second polypeptide chain as shown in the amino acid sequence of SEQ ID NO:21; or Having a first polypeptide chain as shown in the amino acid sequence SEQ ID NO:20, a second polypeptide chain as shown in the amino acid sequence SEQ ID NO:22, a third polypeptide chain as shown in the amino acid sequence SEQ ID NO:23, and a fourth polypeptide chain as shown in the amino acid sequence SEQ ID NO:20; or Having a first polypeptide chain as shown in the amino acid sequence of SEQ ID NO:20, a second polypeptide chain as shown in the amino acid sequence of SEQ ID NO:22, and a third polypeptide chain as shown in the amino acid sequence of SEQ ID NO:24; or It has a first polypeptide chain as shown in the amino acid sequence of SEQ ID NO:20, a second polypeptide chain as shown in the amino acid sequence of SEQ ID NO:22, and a third polypeptide chain as shown in the amino acid sequence of SEQ ID NO:

25.

8. A nucleic acid molecule, characterized in that, The bispecific antibody according to any one of claims 1 to 7 is encoded.

9. An expression vector, characterized in that it comprises the nucleic acid molecule of claim 8; Optionally, the expression vector is a eukaryotic vector or a prokaryotic vector; Optionally, the expression vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

10. A recombinant cell, characterized in that, Carrying the nucleic acid molecule of claim 8 or the expression vector of claim 9; or expressing the bispecific antibody of any one of claims 1 to 7; Optionally, the recombinant cells are obtained by introducing the expression vector of claim 9 into host cells; Optionally, the recombinant cells are prokaryotic cells or eukaryotic cells.

11. A pharmaceutical composition, characterized in that, Includes the multispecific antibody according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9, or the recombinant cell according to claim 10; Optionally, pharmaceutically acceptable excipients may be further included.

12. A reagent kit, characterized in that, It includes the multispecific antibody according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9, or the recombinant cell according to claim 10.

13. Use of the bispecific antibody according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9, or the recombinant cells according to claim 10 in the preparation of a kit for detecting NKp46 and / or HLAG.

14. Use of the bispecific antibody according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9, the recombinant cell according to claim 10, or the pharmaceutical composition according to claim 11 in the preparation of a medicament for the prevention and / or treatment of HLAG-mediated diseases; Optionally, the HLAG-mediated related diseases include cancer; Optionally, the cancer includes at least one of choriocarcinoma, esophageal cancer, head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, bile duct cancer, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

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